Molecular Typing and Epidemiology Profiles of Human Adenovirus Infection among Paediatric Patients with Severe Acute Respiratory Infection in China

Background Human adenoviruses (HAdVs) have been recognised as pathogens that cause a broad spectrum of diseases. The studies on HAdV infection among children with severe acute respiratory infection (SARI) are limited. Objective To investigate the prevalence, epidemiology, and genotype of HAdV among children with SARI in China. Study Design Nasopharyngeal aspirates (NPAs) or induced sputum (IS) was collected from hospitalised children with SARIs in Beijing (representing Northern China; n = 259) and Zhejiang Province (representing Eastern China; n = 293) from 2007 to 2010. The prevalence of HAdV was screened by polymerase chain reaction (PCR), followed by sequence typing of PCR fragments that targeted the second half of the hexon gene. In addition, co-infection with other human respiratory viruses, related epidemiological profiles and clinical presentations were investigated. Results and Conclusions In total, 76 (13.8%) of 552 SARI patients were positive for HAdV, and the infection rates of HAdV in Northern and Eastern China were 20.1% (n = 52) and 8.2% (n = 24), respectively. HAdV co-infection with other respiratory viruses was frequent (infection rates: Northern China, 90.4%; Eastern China, 70.8%). The peak seasons for HAdV-B infection was winter and spring. Additionally, members of multiple species (Human mastadenovirus B, C, D and E) were circulating among paediatric patients with SARI, of which HAdV-B (34/52; 65.4%) and HAdV-C (20/24, 83.3%) were the most predominant in Northern and Eastern China, respectively. These findings provide a benchmark for future epidemiology and prevention strategies for HAdV.

The purpose of this study was to determine the prevalence and genotype (sequencing of the hexon gene after polymerase chain reaction [PCR] screening) of HAdVs among children with SARI in different areas of China from 2007 to 2010. HAdV infections are often associated with the co-infection of bacterial or viral agents [21], frequently leading to severe clinical consequences in hospital patients. Thus, co-infection with other respiratory viruses of HAdV was also investigated.

Ethical approval
This project was approved by the Institutional Review Boards of the Centre of Disease Control and Prevention of China, the Ethical Review Committee of Beijing Hospital, and the Ethics Committee of Wenzhou Medical College. Individual written informed consent was obtained from the parents or guardians of all of the participants.

Study population and specimens
We defined a case of SARI according to the World Health Organization case definition for all hospitalised children in whom the onset of illness occurred within 7 days of admission [22]. Basic demographic and clinical information and laboratory results were recorded for each patient using a standardised form. Specimens were collected on the day of admission. Two separate banks of specimens between May 2007 and March 2010 were used in this investigation. The first set consisted of 259 nasopharyngeal aspirate (NPA) specimens collected from paediatric patients with SARI in Beijing (Northern China). The second specimen bank comprised 293 induced sputum (IS) specimens from paediatric patients with SARI in Zhejiang Province (Eastern China). All of the samples were collected from the patients at the first day of admission and placed in viral transport medium and stored at -80°C until tested.

Laboratory Methods
Nucleic acids (both DNA and RNA) were extracted from 200 μL virus transport medium using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
After the nested-PCR amplification, 5 μL reaction mixtures was subjected to electrophoresis on a 2% agarose gel containing 0.5 μg ethidium bromide per mL. The positive products were gel purified for DNA sequencing using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. DNA sequencing was performed using the Cycle Sequencing Kit (Qiagen) on a DNA analyser.

Phylogenetic tree and typing
The partial hexon gene sequences have been deposited in GenBank under the accession numbers KM377987-KM378038 and KM877524-KM877547. A total of 28 prototype reference sequences of HAdV used for comparisons with sequences from this study were obtained from GenBank (S1 Table). These were aligned and manually adjusted using Clustal X2 and BioEdit. The phylogenetic tree was drawn based on the hexon region of adenovirus by the neighbourjoining (NJ) method using Molecular Evolutionary Genetics Analysis (MEGA) software version 5.0.

Statistical analysis
Data were analysed by the chi-squared test using SAS software version 9.2. P < 0.05 was considered statistically significant.

Demographic and epidemiological data
All of the samples were collected from inpatient children with SARI at hospitals in Beijing (n = 259) and Zhejiang Province (n = 293) from 2007 to 2010. The age and gender distributions are shown in Table 1. The median age of this population is 7 and 7.5 months, respectively. The demographic characteristics of the Northern and Eastern Chinese population were matched well, and no significant differences were observed regarding gender and age distribution (P > 0.05).
In total, 76 (13.8%) of 552 SARI patients were positive for HAdV. The prevalence of HAdV among inpatient children with SARI in Northern China was 20.1% (n = 52), which is higher than that in Eastern China (8.2%, n = 24) (P < 0.0001). Most of the HAdV infections occurred in children aged 6 months to 2 years ( Table 2). The seasonal distribution indicated that most of the HAdV-B infections occurred during the winter and spring (Table 3). However, no season variation was shown for HAdV-C infections in this study.
Recombination is a well-known feature of adenovirus genetics [28,29]. It typically occurs only between strains of the same species [29]; interspecies recombinants are uncommon [20]. It is noteworthy that we found a peculiar HAdV-2/6 cluster in the present study, likely the recombinants of HAdV-2 and HAdV-6. We have amplified the full hexon from one of HAdV-2/ 6 recombinants (GenBank accession No. KP696777). Its position on the phylogenetic tree indicated that the intertypic (HAdV-2/6) recombination occurred in the second half of the hexon gene (data not shown). However, confirmation of this event requires the sequencing of the complete genome.
Previous reports have indicated that HAdV-37 was associated with epidemic keratoconjunctivitis [30,31], and HAdV-57 was isolated from stool specimens of healthy or gastroenteritis-affected children [4,32]. Surprisingly, we found HAdV-37 (KM378014) and HAdV-57 (KM377992) each in single respiratory samples of inpatient children with SARI, suggesting that they may not be limited to the conjunctiva and the gastrointestinal tract.
In conclusion, the present study reported the circulating HAdV types and their prevalence in paediatric patients with SARI in China. HAdV was frequently (76/552, 13.77%) detected, and HAdV-B and HAdV-C were the most predominant in Northern and Eastern China, respectively. However, we only amplified relatively conserved partial hexon sequences rather than the hypervariable region (HVR), perhaps resulting in the loss of the recombinant and variant information. More extensive studies will be needed to address the prevalence and geographic distribution of dominant genotypes of HAdV using well-matched control groups and sequential samples collected over a longer time. Finally, to the best of our knowledge, HAdV-37 (KM378014) and HAdV-57 (KM377992) are the first strains of these genotypes to be reported in inpatient children with SARI.
Supporting Information S1 Table. Reference strain used for phylogenic analysis.